The invention relates to a fed-batch fermentation process which uses special E. coli host/vector systems for the efficient formation of recombinant proteins, in particular recombinant antibody molecules, in particular antibody fragments such as miniantibodies.
Under the conditions according to the invention, the E. coli cells can grow up to very high cell densities at a maximum specific growth rate. After recombinant formation of the product has been switched on, it is only the formed product which has a limiting effect on growth; substrates or metabolic by-products do not limit growth. In this way, and in conjunction with the novel expression vectors which are specially adapted for this purpose and which exhibit high stability, it is possible to achieve high space-time yields of recombinant proteins, which proteins exhibit high biological activity, in particular in the case of antibody fragments.
The culture of E. coli cells to high cell densities is an essential prerequisite for efficient recombinant protein formation. The following cultures are state of the art for this purpose: following unlimited growth (xcexc=xcexcmax) in the batch phase, a carbon source (glucose or glycerol) is customarily metered in, in the subsequent fed-batch phase, in a limited manner such that the formation of growth-inhibiting by-products, for example acetate, is avoided, with the consequence that the growth can be continued in a manner which is only substrate-limited (xcexc less than xcexcmax) until high cell densities are reached (e.g. Riesenberg et al., 1991, J. Biotechnol., vol. 20, 17-28; Strandberg et al., 1994, FEMS Microbiol. Rev., vol. 14, 53-56; Korz et al., 1995, J. Biotechnol. 39, 59-65; EP-B-0 511 226). Growth at a reduced growth rate naturally results in long fermentation times and consequently also in lower space-time yields. Owing to the immediate consumption, the concentration of the carbon source in the culture solution in these fermentations is virtually zero. The substrate-limited conditions are not altered after the recombinant product formation has been switched on.
Fed-batch cultures which use E. coli are also known in which the carbon source is added discontinuously at relatively large time intervals and then in relatively large quantities, with a rise in the pO2 value usually being used as an indicator of substrate exhaustion for the purpose of initiating the subsequent dose of the carbon source (e.g. Eppstein et al., 1989, Biotechnol. 7, 1178-1181). This procedure means frequent switching from relatively long-term substrate excess conditions to substrate limiting conditions and consequently implies metabolic imbalances.
In that which follows, fed-batch cultures are dealt with in which the cells can grow at maximum specific growth rate (xcexc=xcexcmax) in the fed-batch phase. Fed-batch cultures in which relatively large quantities of carbon source are added to the culture at relatively large time intervals, in accordance with off-line determinations, for the purpose of avoiding substrate limitations are experimentally elaborate and suffer from the disadvantage that the concentration of the carbon source is constantly changing during the whole of the fermentation (e.g. Pack et al., 1993, Biotechnol., vol. 11, 1271-1277, Hahm et al., 1994, Appl. Microbiol. Biotechnol. 42, 100-107).
Fed-batch cultures have also been described in which the concentration of the carbon source is measured on-line and is regulated so that limitations are avoided, although these culturesxe2x80x94 in particular in the high cell density regionxe2x80x94 suffer from the disadvantages which are described below. An autoclavable glucose biosensor for use of [sic] microbial fermentations in stirred tank fermenters has recently been described (M. R. Phelps et al., 1995, Biotechnol. Bioeng., vol. 46, 514-524). It was employed for E. coli cultures. This in-situ sensor provides, with a time delay of approximately 2 minutes, the current value in the culture solution. The signal supplied by the glucose sensor is dependent, inter alia, on the pH and pO2. The sensor has not been tested in the high cell density region (X greater than 80 g/l). It is known from experience that growths on in-situ probes when E. coli is used can lead to additional erroneous values at very high cell densities. In addition, it is not possible to recalibrate the sensor exactly during an ongoing fermentation. Instead of being based on measurements using an in-situ sensor, other processes are based, for example, on determining the carbon sources using on-line flow injection analysers (FIA) or on-line HPLC in a culture solution which is removed semi-continuously from the fermenter and rendered cell-free by being subjected to filtration or microcentrifugation (Kleman et al., 1991, Appl. Environ. Microbiol. 57, 910-917 and 918-923; Turner et al., 1994, Biotechnol. Bioeng. 44, 819-829). Prediction and feedback control algorithms have decreased the fluctuations in the glucose concentration during growth up to X=65 g/l (Kleman et al., 1991, Appl. Environ. Microbiol., vol. 57, 910-917). In the region of very high cell densities (from approx. 80 g/l to 150 g/l ), it becomes increasingly more difficult and more time consuming to separate the cells and the nutrient solution, so that the time delay in determining the current glucose value in the fermenter also increases in a biomass-dependent manner and makes it more difficult, or impossible, to maintain the glucose level constant. By contrast, the glucose concentration is measured with a time delay which is constant and brief using an appliance which does without this cell separation (Pfaff et al., 1995, pp. 6-11, in: Proceedings of the 6th International Conference on Computer Appl. in Biotechnol. Garmisch-Partenkirchen, FRG). According to the method of Pfaff et al., an FIA possessing an enzymic-amperometric glucose sensor is employed in the immediate vicinity of the sampling site after the culture has been diluted with a growth inhibitor.
During aerobic culture, E. coli cells which are not obliged by the dosage regime to grow in a sub-strate-limited manner customarily form the metabolic by-product acetate to an increased extent (Riesenberg 1991, Curr. Opinion Biotechnol., vol. 2, 380-384), which acetate accumulates in the nutrient solution and has a growth-inhibitory effect when present in relatively large quantities (Pan et al. 1987, Biotechnol. Lett., vol. 2, 89-94). For this reason, it has only previously been possible to effect fed-batch cultures to high cell densities using special E. coli strains whose accumulation of acetate has been reduced by means of specific genetic alterations, while tolerating other disadvantages which are associated with this. The descendants of E. coli K12 include phosphotransacetylase-negative mutants (Bauer et al., 1990, Appl. Environ. Microbiol., vol. 56, 1296-1302; Hahm et al., 1994, Appl. Microbiol. Biotechnol., vol. 42, 100-107) whose growth is, however, strongly reduced in glucose/mineral salt media. Phelps and collaborators (see above) used the E. coli strain TOPP5 as the host for non-substrate-limited culture up to a biomass of X=85 g/l. However, this E. coli strain, which evidently does not accumulate acetate in a pronounced manner, is not a K12 strain. E. coli TOPP5 forms haemolysin and is consequently a pathogenic strain which is not suitable, for reasons of safety, for use as a host for forming recombinant DNA products in the industrial sector. A reduction in acetate accumulation by means of specifically reorienting the intermediary metabolism was achieved by transforming E. coli cells with a plasmid containing a gene encoding acetolactate synthase (ALS) (San et al., 1994, in: Ann.N.Y.Acad.Sci., vol. 721, 257-267). However, this procedure suffers from the disadvantage that instabilities usually occur under high cell density conditions when an ALS-encoding plasmid is used in combination with a second plasmid carrying the xe2x80x9cproductionxe2x80x9d gene. The efficiency of recombinant product formations is frequently decreased by plasmid instabilities, which occur to an increased extent particularly in association with culture to very high cell densities.
Antibodies or antibody fragments, such as Fabxe2x80x2, F(abxe2x80x2)2, miniantibodies or single-chain Fv""s, are gaining ever increasing importance in the medical and biotechnological spheres. In that which follows, a miniantibody is to be understood, according to the invention, to be a bivalent or bispecific single-chain Fv fragment which is linked by way of a pseudohinge region. In this context, it can be important, for example in cancer therapy, to make available large quantities of antibodies (approximately 1 g/dose). In this respect, monovalent antibody fragments or fusion proteins of these fragments, or multimeric or multispecific variants thereof, can be particularly readily and satisfactorily prepared in E. coli . These fragments or variants are of a small size which is associated with a high specific binding capacity. (E.g. Plxc3xcckthun A., 1992, Immunol. Rev. 130, 151-188; Pack et al., 1995, J. Mol. Biol. 246, 28-34.) However, proteins and antibodies, in particular, must be correctly folded in order to be biologically and functionally active. When considering the yield of formed antibody fragment per cell, attention must be paid to this problem in connection with the cell density. Furthermore, the primary sequence of the antibody is of importance in determining the yield in vitro and the folding in vivo (Knappik A. and Plxc3xcckthun A., 1995, Protein Engin. 8, 81-89). Thus, Fab fragments, for example, are expressed as insoluble cytoplasmic or periplasmic aggregates and refolded in vitro. Thus, yields of about 0.14 g/l at low cell density (Condra et al., 1990, J. Biol. Chem. 265, 2292-2295) and of up to about 1-2 g/l of insoluble antibodies at medium cell density (Shibui et al., 1993, Appl. Microbiol. Biotechnol. 38, 770-775) have been reported. Bivalent miniantibodies (Pack et al., 1993, Biotechnol. 11, 1993, 1271-1277) can also be obtained in E. coli in biologically functional form in yields of about 0.2 g/l. On average, approximately 5-45% of these yields is properly refolded.
In the known E. coli systems, the formation of foreign protein is, as a rule, switched on in a suitable manner, after appropriate cell densities have been reached, by a regulatable promoter system corresponding to the expression system. Examples of promoter systems which may be mentioned here are (i) the araBAD promoter in the presence of the AraC repressor (inducible by arabinose) (e.g. Better et al., 1993, Proc. Natl. Acad. Sci. (USA) 90, 457-461), (ii) the phoA promoter (inducible by withdrawing phosphate) (e.g. Carter et al., 1992, Biotechnol. 10, 163-167) and (iii) the lac promoter system (inducible by IPTG) (Pack et al., 1993, loc. cit.) . While the lac system brings about good expression as a rule, it suffers from the disadvantage that, on the one hand, undesirable basal expression is observed prior to induction of the promoter and, on the other, plasmid instability is observed following induction with ITPG [sic].
In a particular embodiment of the invention, a special vector (pHKK) is described which contains, as the foreign gene, sequences which encode fragments of the murine or humanized antibody Mab 425. Mab 425 (ATCC HB 9629) is a murine monoclonal antibody which was isolated from the known human A432 cancer cell line (ATCC CRL 1555) and binds to the epitope of human epidermal growth factor receptor (EGFR, a glycoprotein of about 170 kD) while inhibiting the binding of the natural ligand EGF. It has been demonstrated that Mab 425 has a cytotoxic effect on tumour cells or is able to impair the growth of these cells (Rodeck et al., Cancer Res. 1987, 47: 3692). WO 92/15683 discloses humanized and chimeric forms of Mab 425, including the DNA and amino acid sequences of their light and heavy chains.
The object of the invention was to make available a process for preparing foreign proteins, in particular antibody fragments, in recombinant E. coli cells under high cell density conditions (HCDC=high cell density culture) with high space-time yields, and without any substantial impairment of growth by substrates or metabolites and without significant plasmid losses or plasmid instabilities, while ensuring that the expressed protein exhibits a high degree of effective biological activity (binding capacity and correct folding).
The process according to the invention is a multi-step batch process which is primarily notable for the fact that the cells are able to grow at a maximum rate during the whole of the batch (xcexc=xcexcmax). Thus, cell densities of from 100 to 150 g/l (bio dry mass [sic]) can ultimately be achieved using the described process. Furthermore, the growth is not inhibited to an important extent by acetate accumulation since, surprisingly, such an accumulation is not particularly pronounced under the conditions which are selected, in particular when E. coli strains are used which in any case only tend to form decreased quantities of acetate during the fermentation. This is achieved by, also in association with a series of other additional measures, first and foremost, in the fed-batch phase which is inserted after a batch phase, keeping the concentration of the carbon source in the medium constant in a defined range while maintaining unlimited growth of the cells. By designing the relevant expression vector in an appropriate manner, the undesirable basal expression of protein, prior to switching on protein synthesis by means of a regulatable promoter system, can also be virtually eliminated, as can the plasmid loss, which is sometimes substantial and which, as already mentioned above, can normally be observed in expression systems using strong promoters such as the lac promoter system.
Protein yields of on average from 3 to 5 g/l can be achieved after a total culture time of from approx. 25 to 35 hours. In the case of the antibody fragments, in particular miniantibodies, which are particularly critical owing to their folding criteria, approximately 80% of the synthesized material is biologically active and correctly folded.
The invention consequently relates to a process for preparing foreign protein, in E. coli cells which have been transformed with a plasmid carrying the foreign gene and an inducible promoter, by means of high cell density fermentation by way of batch and fed-batch stages, without any restriction of growth by sub-strates or metabolic by-products, and isolation and purification of the expressed protein from the culture medium, with the concentration of substrates in the fed-batch phase being controlled using a continuous, automated or semi-automated analysis and addition system, with, in the fed-batch phase, (i) the concentration of the carbon source in the medium being kept constant in a range between 0.1 g/l and 25 g/l while maintaining unlimited growth of the cells (xcexc=xcexcmax), (ii) the production of the foreign protein being started in the said fed-batch phase by inducing the promoter at a cell density of between 10 and 80 g/l, and (iii) utilizable nitrogen and phosphate, and also salts of trace elements, being fed in continuously after induction of product synthesis has taken place, where (iv) the pO2 value is adjusted to between 5 and 25% during the whole of the fed-batch phase by passing oxygen into the fermentation broth in an appropriate manner.
The values according to the invention for the requisite concentration of the carbon source during the fed-batch phase are in a range from 0.1 g to 25 g/l. A preferred range is between 0.5 and 15 g/l, in particular from 1.0 to 5 g/l, or from 1.0 to 3 g/l. The particularly preferred concentration is 1.5 g/l. Preferred carbon sources which may be mentioned are glucose or glycerol or mixtures of these two compounds. According to the invention, the carbon source is added in a continuous manner (on-line) using an automated or semi-automated addition and analysis system. An on-line flow injection analysis system (FIA) is preferably employed.
The feeding-in of utilizable nitrogen, preferably ammonium nitrogen and phosphate, for example diammonium hydrogen phosphate or ammonium dihydrogen phosphate, and also trace elements, for example salts of boron, manganese, copper, molybdenum, cobalt, iron or zinc which are soluble in the medium, takes place in the fed-batch phase which is inserted after the batch phase, preferably after switching on protein synthesis using the regulatable promoter, at a cell density of from 50 to 80 g/l (bio dry mass [sic]), preferably at about 70 g/l, at a total growth rate [sic] of 100 to 150, preferably 140, g/l.
According to the invention, protein synthesis is switched on, by activating the regulatable promoter system, at a cell density of from 10 to 80 g/l, preferably from 20 to 60 g/l; very particularly preferably, the range is from 40 to 50 g/l.
During the fed-batch phase, the partial pressure of oxygen is between 5 and 25%, preferably between 15 and 25%, very particularly preferably 20%.
According to the invention, the pH of the fermentation medium has to be adjusted, during the whole batch, to between 6.5 and 7.0, preferably to between 6.7 and 6.9, in particular to 6.8.
The invention furthermore relates to a corresponding process in which an expression vector is employed which possesses an expression cassette which contained [sic] the foreign gene and is flanked by two terminator sequences. These terminator sequences, in particular that which is positioned upstream, successfully prevent unwanted expression of protein prior to the expression being switched on by the promoter system. While the terminator thp (Nohno et al., 1988, J. Bacteriol. 170, 4097-4102) is particularly suitable, other known terminator sequences may also be employed.
The invention furthermore relates to a process in which the expression vector which is employed additionally contains a suicide system. The suicide system produces a protein which is toxic for the cell if the plasmid is not present in the cell. Suitable suicide systems are known from the literature. A suicide system which is particularly suitable for the invention is the hok-sok system (e.g. Gerdes K., 1988, Biotechnol. 6, 1402-1405). Thus, it is important, for the process for effectively forming recombinant proteins, in particular antibody molecules, that the host/vector system is characterized, in the high cell density region, by high plasmid stability, low recombinant basal expression and high product formation. In this context, suicide systems, in combination with recombinant expression cassettes which are flanked by terminators, are vector-specific.
The invention furthermore relates to a corresponding process in which a foreign gene is employed which encodes an antibody fragment, in particular a miniantibody.
The invention also relates to a process in which expression vectors are employed which possess additional features which are described below.
In principle, most of the E. coli strains can be employed which are known and which are suitable for recombination technology and for production on an industrial scale. Advantageously, those strains are preferably used which accumulate relatively little acetate during growth to high cell densities. Those strains are particularly suitable which exhibit an acetate enrichment of less than 5 g/l. Surprisingly, the acetate accumulation can be kept particularly low by using the chosen conditions of the process according to the invention. The well known and commercially available E. coli strain RV308 (ACCC [sic] 31608), and its variants having the same effect, is particularly suitable in this regard.
The invention therefore relates, in particular, to a corresponding process in which an E. coli strain is employed which exhibits an acetate accumulation of less than 5 g/l in the culture medium during the fermentation.
The invention also relates to an E. coli expression vector which is suitable for expressing foreign proteins under high cell density fermentation conditions and which exhibits the following features:
and, in a preferred embodiment, also a suicide system, in particular the hok-sok suicide system.
According to the invention, the promoter system can also be replaced by other suitable systems, for example those mentioned above. Likewise, other signal sequences and control sequences having the same effect are also encompassed by the invention.
Finally, the invention relates to the expression vector pHKK (FIG. 2), which is defined by its construction and which contains the sequences for the miniantibody which is derived from Mab 425, and to a special recombinant E. coli host RV308[pHKK], as special embodiments.